Methods of generating a pegylated il-11 composition

ABSTRACT

Methods are provided for producing a PEGylated interleukin 11 (IL-11) tby treating a recombinant IL-11 PEGylated with an equimolar to low molar excess of PEG carrying an amine-reactive group to achieve a highly pure monoconjugate preparation, which provides improved half-life in serum while having desirable therapeutic activity and presenting fewer side-effects. Most preferably, the IL-11 is an N-terminally truncated human or humanized IL-11 and has a 20 Kd or 40 Kd branched PEG moiety, Y- or comb-shaped in particular, coupled to the N-terminal amino group. Such compounds are characterized by substantially increased stability in serum and sustained biological activity while exhibiting significantly reduced plasma expansion.

This application is a continuation of U.S. patent application Ser. No.16/418,850, filed May 21, 2019, which is a divisional application ofU.S. patent application Ser. No. 15/554,415, filed Aug. 29, 2017.

FIELD OF THE INVENTION

The field of the invention is pharmaceutical compositions and methods,especially as they relate to PEGylated Interleukin 11 (IL-11).

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art. All publications herein areincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Where a definition or use ofa term in an incorporated reference is inconsistent or contrary to thedefinition of that term provided herein, the definition of that termprovided herein applies and the definition of that term in the referencedoes not apply.

Chemotherapy-induced thrombocytopenia remains an unmet medical needbecause the current treating regimen employs transfusion of plateletsthat may be in short supply and carry a risk of viral contamination. Onthe other hand, recombinant human IL-11 can be given to a patient tostimulate platelet production. However IL-11 administration requiresdaily dosing, leading to marginal clinical efficacy and plasmaexpansion.

IL-11 is a cytokine and acts as a major signaling agent inhematopoiesis, and especially in the stimulation of megakaryocytematuration. Action of IL-11 is typically mediated by the IL-11 receptorand glycoprotein gp130 with subsequent phosphorylation/activation ofgp130. Clinical uses for IL-11 include treatment of side effectsassociated with chemotherapy, which is thought to enhancemegakaryocytopoiesis and increase platelet counts. Recombinant humanIL-11 is commercially available as NEUMEGA® (Oprelvekin, Wyeth-Ayerst)and is approved for the prevention of severe thrombocytopenia and thereduction of the need for platelet transfusions followingmyelosuppressive chemotherapy in adult patients with non-myeloidmalignancies who are at high risk of severe thrombocytopenia. NEUMEGA®is typically supplied in a single use vial containing 5 mg IL-11 as alyophilized powder for reconstitution with 1 mL sterile water forinjection (administered at a dose of 25-50 μg/kg/day). The most frequentadverse event associated with NEUMEGA™ is plasma expansion leading tolife-threatening atrial arrhythmias, syncope, dyspnea, congestive heartfailure, and pulmonary edema.

IL-11 is cleared from the circulatory system relatively quickly and assuch requires multiple injections. For example, Neumega™ subcutaneouslyadministrated in healthy men has a terminal half-life about 6.9 hours(Product Insert of Neumega™). The poor pharmacokinetics such as rapidrenal excretion and proteolytic digestion, as well as its associatedadverse effect often reduce clinical prevalence. Moreover, dailyinjection also means hospitalization to manage adverse event that notonly adds on medical expense but also damages quality of life topatients. As a result, platelet transfusion remains the gold-standardfor treating chemotherapy-induced thrombocytopenia (CIT).

Several attempts have been undertaken in the art to increase serumstability while maintaining beneficial therapeutic potential of suchcompositions. For example, US 2010/0098658 reports an IL-11 analog(mIL-11) in association with a polymer (PEG) that exhibited enhancedresistance to acidolysis and increased serum half-life. In anotherattempt to stabilize IL-11, as described in U.S. Pat. No. 8,133,480,cysteine variants of IL-11 were prepared and selected muteins werefurther modified with PEG to increase serum stability. While thesemodifications have improved serum stability or half-life of IL-11 to atleast some degree, one or more disadvantages nevertheless remain,including marginal efficacy in myelosuppressive animals, complexity inproduction, repeated dosing, and formulation into injectable solution.

Because of lacking cysteine residues in IL-11, the '480 patent describesinsertion of a cysteine residue in the C-terminal amino acid sequence,conferring a functional group to allow conjugation of a thiol-reactivepolyethylene glycol chain. Although the biological activity wasconserved, the introduction of cysteine may yield intermolecular dimersand the production yield of insect cells may be lower than bacterialproduction. Additionally the serum half-life of so modified IL-11 whenadministrated intravenously in male Sprague-Dawley rats was about 5.6 hrfor 40 KD PEGylation, which is less than desirable. Moreover, in animalstudies using cyclophosphamide-treated rats, the efficacy was marginalwith the-other-day dosing scheme. Another employed PEG conjugation ontoN-terminally truncated sequence of IL-11 with 20 KD PEG via amine oramide bonding was described in US 2010/0098658. Although the N-terminaltruncation did not reduce its biological activity, the serum half-lifeadministered subcutaneously in male Sprague-Dawley rats was about 8.5hr, again falling short of desirable stability. Additionally, theefficacy in an animal disease model was unknown.

Linear or branched PEG of 20 KD conjugating onto amine groups of IL-11was reported (Takagi et al. 2007, “Enhanced pharmacological activity ofrecombinant human interleukin-11 (rhIL11) by chemical modification withpolyethylene glycol.” J Control Release, 119(3):271-278), suchunspecific conjugation often resulted in multiple PEGylation viareaction with lysine, histidine, and tyrosine residues as well asN-terminal amines.

Other reports have demonstrated certain carbohydrate modifications onthe “non-core” regions of IL-11 such as N-terminus and loops enhancedcell-stimulatory activities, suggesting these regions are perhapsdesigned to limit biological activity of IL-11 (Yanaka et al. 2011,“Non-core region modulates interleukin-11 signaling activity: generationof agonist and antagonist variants.” J. Biol. Chem., 286:8085-8093).However, no desirable modification was reported with stabilities andactivities above unmodified IL-11.

Thus, even though several methods of stabilizing IL-11 are known in theart, all or almost all have one or more drawbacks, such as limitedefficacy and requirement for repeated dosing. More importantly, even inmodified form, adverse effects of IL-11 (e.g., plasma expansion) werenot reduced. Therefore, there remains a need for improved compositionsand methods to stabilize IL-11 while simultaneously alleviate adverseeffects.

SUMMARY OF THE INVENTION

The inventive subject matter is directed to compounds, compositions, andmethods for improving stability and half-life time of IL-11 in serumwhile maintaining biological activity and mitigating side-effects. Inespecially preferred aspects, the inventors have discovered that theamino acid position, manner of attachment, and type of PEG is criticalto producing stable and biologically active PEGylated IL-11, andparticularly preferred PEGylated IL-11 will have the same sequence asnative human IL-11 but lack the N-terminal first amino acid, proline.Moreover, such IL-11 will preferably be covalently modified at theN-terminus with a possible secondary site at certain lysine residueswithin the polypeptide chain. Most typically, the average molar ratio ofIL-11 to PEG compound attached to the IL-11 is 1:1.

In one aspect of the inventive subject matter, the inventors contemplatea modified interleukin 11 (IL-11) compound that includes an IL-11polypeptide chain that is covalently coupled to a PEG moiety, whereinthe PEG moiety has an average molecular weight of between 10-50 Kd andhas distinct first and second PEG portions, wherein the PEG moiety iscovalently bound to an N-terminal amino acid, and wherein the IL-11polypeptide chain is a human or humanized polypeptide chain.

Most typically, the IL-11 polypeptide chain is a human IL-11 polypeptidechain, and/or maybe shortened by deletion of an N-terminal proline. Forexample, especially suitable IL-11 polypeptide chain may have a sequenceaccording to SEQ ID NO:1. With respect to the PEG moiety it is generallypreferred that the moiety has an average molecular weight of 20 Kd or 40Kd, and/or that the PEG moiety has a Y shape. While not limiting to theinventive subject matter, it is preferred that the molar ratio ofpolypeptide chain to PEG moiety is about 1:1 (e.g., 0.9:1 to 1:0.9, or0.8:1 to 1:0.8). In addition, it is contemplated that a second PEGmoiety may be covalently coupled to the modified IL-11 via an aminogroup of the IL-11 polypeptide chain. Furthermore, it is generallypreferred that the PEG moiety is covalently bound to the N-terminalamino acid via an amine bond (however, amide bonds are also specificallycontemplated).

Viewed from another perspective, the inventors also contemplate apharmaceutical composition that include a therapeutically effectiveamount of an IL-11 compound according to the inventive subject matter(e.g., as described above), in combination with a pharmaceuticallyacceptable carrier. Where desirable, the composition may be formulatedfor injection, and may include the IL-11 compound is present in anamount to provide a dosage unit of between 10-100 μg/kg for a pediatricor adult patient. Additionally, it is contemplated that the compositionmay be lyophilized, or in a liquid form for injection or infusion. Asbest suitable, the pharmaceutical composition may further include asecond pharmaceutically active compound, separately, or in admixturewith the IL-11 compound. Thus, kits comprising contemplatedpharmaceutical compositions together with other components (e.g., secondpharmaceutically active compound such as a steroid, an agent thatstimulates platelet production in bone marrow, an antibody, ananalgesic, or anti-inflammatory agent, or a solvent for reconstitution)are also expressly contemplated herein

Consequently, the inventors also contemplate use of an IL-11 compoundaccording to the inventive subject matter in the manufacture of apharmaceutical composition. While not limiting to the inventive subjectmatter, especially contemplated treatments include (a) nuclearaccident/radiation induced bone and gastrointestinal damage; (b)chemotherapy induced bone and gastrointestinal damage; (c) burn inducedthrombocytopenia and gastrointestinal damage; (d) chemotherapy inducedthrombocytopenia; (e) trauma-, cancer-, or infection-inducedgastrointestinal damage or inflammatory bowel disease, (f) freeradical-induced lung damage, and (g) cardiovascular diseases. As notedbefore, it is generally contemplated that the pharmaceutical compositionis formulated for injection and/or that the pharmaceutical compositionis lyophilized.

In a further aspect of the inventive subject matter, the inventorstherefore also contemplate a method of increasing serum half-life of aninterleukin 11 (IL-11) compound. Preferred methods will include a stepof covalently coupling an IL-11 polypeptide chain to a PEG moiety,wherein the PEG moiety has an average molecular weight of between 10-50Kd and has distinct first and second PEG portions, wherein the PEGmoiety is covalently bound to an N-terminal amino acid, and wherein theIL-11 polypeptide chain is a human or humanized polypeptide chain. Mosttypically, the IL-11 polypeptide chain is a human IL-11 polypeptidechain, and/or the IL-11 polypeptide chain is shortened by deletion of anN-terminal proline (e.g., having a sequence according to SEQ ID NO:1).

In further contemplated methods, the PEG moiety has an average molecularweight of 20 Kd or 40 Kd, and/or may have a Y shape. Where desired, themolar ratio of the polypeptide chain to the PEG moiety is about 1:1, andit is further contemplated that the methods may further include a stepof covalently coupling a second PEG moiety via an amino group in theIL-11 polypeptide chain. As before, it is contemplated that the PEGmoiety is covalently bound to the N-terminal amino acid via an aminebond.

In further contemplated methods, the inventors contemplate a method oftreating a condition that is responsive to administration of IL-11. Suchmethods will typically include a step of administering contemplatedpharmaceutical compositions in a therapeutically effective amount to apatient in need thereof. For example, suitable condition may be selectedfrom the group consisting of (a) nuclear accident/radiation induced boneand gastrointestinal damage; (b) chemotherapy induced bone andgastrointestinal damage; (c) burn induced thrombocytopenia andgastrointestinal damage; (d) chemotherapy induced thrombocytopenia; (e)trauma-, cancer-, or infection-induced gastrointestinal damage orinflammatory bowel disease, (f) free radical-induced lung damage, and(g) a cardiovascular disease. Exemplary preferred pharmaceuticalcomposition for these methods may comprise IL-11 I40NY or I20NY, and itis further contemplated that IL-11 is administered (e.g.,subcutaneously) in a dosage between 10-100 μg/kg.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the primary sequence of IL-11 without N-terminal proline.

FIG. 2 are images of SDS-PAGE gels with molecular weight markers andvarious PEGylated forms of IL-11 as indicated.

FIG. 3 is a graph depicting plasma concentrations of various IL-11compositions after single i.v. administration.

FIG. 4 is a graph depicting platelet increment after single i.v.administration of various IL-11 compositions.

FIG. 5 depicts chromatograms of peptide maps for tryptic digestion ofunconjugated IL-11, I40NY and I40KY in comparison.

FIG. 6 is a graph depicting platelet increment after s.c. administrationof various IL-11 compositions (Daily injection for IL-11 in consecutive14 days; and weekly injection for PEGylated counterparts).

FIG. 7 is a graph depicting hematocrit reduction after s.c.administration of various IL-11 compositions (Daily injection for IL-11in consecutive 14 days; and weekly injection for PEGylatedcounterparts).

FIG. 8 is a graph suggesting a correlation between maximal plateletinduction and maximal reduction of hematocrit.

FIG. 9 are graphs depicting cell proliferation activity of PEGylatedcompounds in 7TD1 assays, in comparison to unconjugated IL-11.

FIG. 10 is an image of a non-reducing SDS-PAGE gel with silver stainillustrating the purity of I40NY at various loading quantities.

FIG. 11 is a HPLC chromatogram depicting the product purity ofmono-PEGylated component for I40NY.

FIG. 12 is a pharmacokinetic profile depicting the kinetics of theplasma concentration of I40NY after single s.c. administration, incomparison to single s.c. administration of unconjugated IL-11.

FIG. 13 is an overlay of circular dichroism spectra of IL-11 and I40NY.

FIG. 14 is an ellipticity plot for IL-11 and I40NY as a function oftemperature.

FIG. 15 is a pharmacodynamics profile depicting the platelet productionof contemplated compounds in an animal model of myelosuppressive rats.

FIG. 16 is a graph depicting the hematocrit reduction of contemplatedcompounds in an animal model of myelosuppressive rats.

DETAILED DESCRIPTION

The inventors have discovered that the type of PEG compound, thelocation of covalent attachment, and the primary sequence of IL-11 aredeterminants to the stability and activity of so modified IL-11. Inparticularly preferred and unexpected aspects, the inventors discoveredthat IL-11 has substantially improved stability when truncated at theN-terminus by one amino acid that is then PEGylated. Furthermore, theinventors also discovered that the particular type and molecular weightof the PEG moiety is an additional determinant of stability, activity,and toxicity as is further described in more detail below.

Contemplated Compounds

To investigate the influence of type, molecular weight, and attachmentposition of PEG to IL-11, the inventors prepared various PEGylated IL-11molecules from recombinant human IL-11 having a primary sequence asshown in FIG. 1 (identical with the native human IL-11 sequence, butlacking the N-terminal proline). It is generally preferred that theIL-11 protein is a N-terminal truncated or modified human IL-11. Forexample, especially preferred truncated forms include IL-11 moleculesthat lack at least one or two or three (or more) N-terminal amino acids.Alternatively, the IL-11 may also be modified to have a N-terminal aminoacid that is different from the human unmodified counterpart. Forexample, a modified IL-11 may lack the first N-terminal amino acid andmay have a second amino acid that is other than the second amino acidfound in unmodified human IL-11 (e.g., lacking P and having G replacedby V). Most typically, N-terminal amino acids will be stabilizing aminoacids and therefore especially include M, G, A, S, T, V, or P, and infurther contemplated aspects, destabilizing amino acids (e.g., F, Q, N,R, etc.) may be replaced by stabilizing amino acids. Deletions of one ormore amino acids from the N-terminal end will typically be limited tothe first ten, or the first five, or the first three amino acids. On theother hand, in less preferred aspects, deletions of one, two, three,four, five, or more amino acids may also be implemented at theC-terminus of the IL-11 moiety. As a general guidance, deletions willgenerally be limited to those that do not or only moderately adverselyaffect biological activity and/or stability (e.g., loss of activityand/or stability is less than 20%, and more typically less than 10%).Alternatively, or additionally, contemplated IL-11 molecules alsoinclude fusion proteins with IL-11 an exemplary fusion proteins includethose described in US 2010/0143973, which is incorporated by referenceherein. Most typically, the IL-11 is a recombinant protein and may beexpressed in a suitable expression system, and most preferably in aprokaryotic system (e.g., E. coli) or yeast system (e.g., Pichiapasteuris). Of course, it should also be recognized that particularlypreferred forms of IL-11 are mature forms (i.e., without leadersequence)

Moreover, it should be appreciated that suitable IL-11 molecules neednot be human IL-11 but may be of any other (typically mammalian) origin.Therefore, suitable IL-11 sources (recombinant or native) includeprimate, murine, porcine, equine, etc. These sequences may then be atleast partially humanized to reduce immunogenicity and/or increasestability and/or activity in human. Similarly, synthetic consensussequences are also contemplated herein.

PEGylation of contemplated IL-11 molecules may be performed in numerousmanners and includes covalent as well as non-covalent methods. However,it is generally preferred that the PEGylation uses covalent binding tothe IL-11. There are numerous manners known in the art to covalentlyattach a PEG group to a protein and suitable methods include those thatreact the N-terminal amino group or the C-terminal carboxylic acid groupwith a suitable reactive group on the PEG moiety (e.g., aldehyde,maleimide, acid chloride, etc.), or sulfhydryl reactive groups (e.g.,maleimide, pyridyl disulfide, vinyl sulfone, etc.) that allow fordisulfide bonding to cysteine groups, or amino reactive reagents thatreact with an ε-amino group of a lysine amino acid (e.g., NHS-esters,NHS-carbonates, triazine, groups, etc). Therefore, it is alsocontemplated that one or more amino acids may be added to the N- and/orC-terminus to introduce a reactive group suitable for attachment of aPEGylation group. For example, serine or threonine may be added to allowfor enzymatic attachment using a N-actylgalactosamine or PEG sialicderivative, or a lysine for covalent attachment to the ε-amino group, ora phenyl alanine or threonine group for attachment to a hydroxyl group.

With respect to suitable PEG molecules for use herein, it is generallycontemplated that various molecular weights for PEG are appropriate, andcontemplated molecular weights are between 2 Kd and 200 Kd (average ornominal molecular weight). However, particularly preferred molecularweights (average or nominal molecular weight) include those between10-50 Kd per linear chain in a PEGylation moiety. Moreover, it isgenerally preferred that the PEG moiety will have a single linear, orY-shaped PEG moiety, and even more preferably that such PEG moiety willhave a molecular weight of between 20-40 Kd. Alternatively, suitable PEGmoieties may also include dendrimeric PEG constructs, as well as PEGmoieties with more than two linear chains. Where the PEG moieties havemore than one linear PEG chain, it is generally preferred that thechains have an average molecular weight that is substantially the same(average molecular weight difference less than 15%).

In further preferred aspects, the PEG moiety is covalently attached tothe IL-11 via the N-terminal amino group of IL-11 and/or (optionally) toan ε-amino group of an internal lysine or the ring nitrogen ofhistidine. Due to the N-terminal covalent bond, it is preferred that themolar ratio of IL-11 to PEG moiety is about 1:1 (e.g., 0.9:1 to 1:0.9,or 0.8:1 to 1:0.8, etc.). In addition, it should be appreciated thatmoderate levels of PEGylation may be present at internal amino acidresidues (e.g. between 10%-20%, or between 1%-10% of all IL-11 may carryan extra PEGylated internal amino acid). For example, a second PEGmoiety may be attached to an ε-amino group of an internal lysine orhistidine. As is further shown in more detail below, a particularlypreferred form of PEGylated IL-11 is I40NY, comprising human IL-11(lacking N-terminal proline), to which is attached on the N-terminus aY-shaped PEG moiety with an average molecular weight of 40 Kd.

In still further alternative aspects, it should be appreciated that thePEGylation may be mixed with respect to the attachment position of thePEG moiety and/or kind of attachment. Therefore, IL-11 may be subjectedto random non-covalent PEGylation and site specific PEGylation at anN-terminal amino acid, or subjected to different site specificPEGylations at the N-terminal amino acid and an internal amino acid. Forexample, and most preferably, IL-11 (or any modified form thereof) maybe PEGylated at the N-terminal amino acid and optionally at an internalamino acid via a nitrogen atom (e.g., from lysine or histidine) inaddition to the N-terminal modification.

For example, and using the truncated IL-11 as shown FIG. 1, theinventors performed PEGylation using the PEG reagents as shown in Table1 (where n and m are independently an integer between 80 and 1000,depending on the molecular weight of the compound) followingexperimental protocol as provided by the manufacturer and as describedin more detail further below.

TABLE 1 Manufacturer/ Cat. No. Structure/Molecular size NOF/SUNBRIGHTME-200AL

Jenkem/Y-PLAD-40K

NOF/SUNBRIGHT ME-120TS

NOF/SUNBRIGHT GL2-4000TS

NOF/SUNBRIGHT GL2-200AL3

NOF/SUNBRIGHT ME-050TS

NOF/SUNBRIGHT GL4-400AL3

After PEGylation of the truncated IL-11, the so obtained compounds werepurified as also addressed in more detail below, and the variousPEGylated IL-11 molecules had the following designations as shown inTable 2:

TABLE 2 Compound PEG size/ Structure code KDa of PEG Conjugation site(bonding) I20NL Single 20 linear N-terminal (amine bond) I40NY Single 40Y-shaped N-terminal (amine bond) I12KL Single 12 linear N-terminal(amide bond) I40KY Single 40 Y-shaped N-terminal (amide bond) I20NYSingle 20 Y-shaped N-terminal (amine bond) I20NL2 20 × 1^(~)3 linearN-terminal/lysine or histidine (amine bond) I20NY2 20 × 1^(~)3 Y-shapedN-terminal/lysine or histidine (amine bond) I05KL4  5 × 4^(~)5 LinearN-terminal/lysine/ histidine (amide bond) I40NX Single 40 4-armN-terminal (amine bond)

Most notably, the inventors have discovered that the type of PEG moietyand the site of attachment (and to some degree the sequence of theIL-11) had unexpected and substantial influence on biological activityand stability in vivo. As is more evident from the experimental databelow, especially preferred PEGylations are at the N-terminal amino acidusing a single Y-shaped PEG moiety, particularly where the IL-11 wastruncated.

Contemplated Compositions

Based on the inventors' discovery of extended biological activity ofcontemplated compounds, it is generally contemplated that the compoundsaccording to the inventive subject matter may be formulated fortreatment of various diseases associated with a lack of IL-11 orcharacterized by a therapeutic response to treatment with IL-11.Therefore, and among other contemplated uses, the inventors especiallycontemplate that pharmaceutical compositions comprising contemplatedcompounds may be effective for the treatment or prevention of (a)chemotherapy-induced thrombocytopenia, (b) nuclear accident/radiationinduced bone and gastrointestinal (GI) damage; (c) chemotherapy inducedbone and GI damage; (d) burn induced thrombocytopenia and GI damage; (e)other causes of thrombocytopenia; (f) other causes of GI damage,including inflammatory bowel diseases like Crohn's Disease andulcerative colitis, as well as pseudomembraneous colitis, (g) freeradical-induced lung damage, and/or (h) cardiovascular diseases, whereincontemplated pharmaceutical compositions comprise a therapeuticallyeffective amount of contemplated compounds (or pharmaceuticallyacceptable salt, hydrate, or prodrug thereof), and a pharmaceuticallyacceptable carrier. For example, in one aspect of the inventive subjectmatter, contemplated compositions are formulated for treatment ofchemotherapy-induced thrombocytopenia or GI damage or radiation inducedbone and gastrointestinal (GI) damage. Alternatively, or additionally,it should also be appreciated that contemplated compositions may beformulated to induce acute phase proteins, and/or to modulateantigen-antibody responses.

It is particularly preferred that contemplated compounds are included ina composition that is formulated with one or more non-toxicpharmaceutically acceptable carriers. Suitable pharmaceuticalcompositions are preferably formulated for injection or infusion, or fororal administration in solid or liquid form. Thus, it should beappreciated that pharmaceutical compositions according to the inventivesubject matter may be administered to humans and other (typicallymammalian) animals using various routes, including parenterally, orally,intraperitoneally, and topically.

For example, suitable pharmaceutical compositions for injectionpreferably comprise pharmaceutically acceptable sterile aqueous ornonaqueous solutions, dispersions, emulsions, or suspensions, as well assterile powders for reconstitution into sterile injectable solutions ordispersions prior to use. Examples of suitable aqueous and nonaqueouscarriers, diluents, solvents, or vehicles include water, Ringer'ssolution, and isotonic sodium chloride solution, ethanol, polyols (e.g.,glycerol, propylene glycol, polyethylene glycol, etc.), and suitablemixtures thereof, oils, and injectable organic esters (e.g., ethyloleate). Contemplated compositions may also contain various inactiveingredients, including preservatives, wetting agents, emulsifyingagents, and/or dispersing agents. Sterility may be ensured by inclusionof antibacterial and/or antifungal agents (e.g., paraben, phenol sorbicacid, chlorobutanol, etc.), as well as by filtration across sub-micronmembranes (e.g., 0.45 μM or 0.22 μM pore size), autoclaving orpasteurizing, and radiation (e.g., gamma or e-beam). Where appropriate,osmotically active agents may be included (e.g., sugars, sodiumchloride, etc.). While not limiting to the inventive subject matter,contemplated formulations for injection are typically in a pH range of3-9, more typically 6-8, and most typically 7.4+/−0.3. Of course, itshould also be recognized that all liquid formulations may be preservedin various manners to facilitate long-term storage/stockpiling. Forexample, contemplated manners of stabilization include water/solventremoval using lyophilization, spray-drying, crystallization, adsorptionon (preferably biocompatible or pharmaceutically acceptable) solidphases, etc.

The compositions according to the inventive subject matter may beadministered using various routes, including orally, parenterally, byinhalation, topically, rectally, nasally, or via an implanted reservoir,wherein the term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrathecal,intrahepatic, intralesional, and intracranial administration (typicallyinjection or infusion). Preferably, the compositions are administeredvia injection, typically intravenously, and more preferablysubcutaneously. Contemplated pharmaceutical compositions may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, the lower intestinal tract, or areasexposed during surgical intervention. There are numerous topicalformulations known in the art, and all of such formulations are deemedsuitable for use herein.

With respect to the amount of contemplated compounds in the composition,it should be recognized that the particular quantity will typicallydepend on the specific formulation and desired purpose. Therefore, itshould be recognized that the amount of contemplated compounds will varysignificantly. However, it is generally preferred that the compounds arepresent in a minimum amount effective to deliver a therapeutic effect invitro and/or in vivo.

Thus, in most preferred embodiments, contemplated compounds will bepresent in an amount of between about 0.1 μg/ml to about 100 mg/ml, moretypically in an amount of between about 10 μg/ml to about 10 mg/ml, andmost typically between about 5 μg/ml to about 100 μg/ml. With respect toa dosage unit, it is generally contemplated that contemplated compoundsare administered at a dosage effective to achieve a desired therapeuticeffect, typically 10-100 μg/kg, and more preferably 30-70 μg/kg.However, alternate dosage units may be between 0.1-10 μg/kg, or 50-80μg/kg, or 80-120 μg/kg, or 120-200 μg/kg, or even higher. Viewed from adifferent perspective, it should be appreciated that a single-use unitof contemplated formulations may include between about 0.3 mg to 3.0 mgof PEGylated IL-11, or between about 3 mg to 7 mg of PEGylated IL-11, orbetween about 7 mg to 10 mg of PEGylated IL-11 (most typically with aspecific activity of 7-9×10⁶ U/mg). Unless the context dictates thecontrary, all ranges set forth herein should be interpreted as beinginclusive of their endpoints, and open-ended ranges should beinterpreted to include commercially practical values. Similarly, alllists of values should be considered as inclusive of intermediate valuesunless the context indicates the contrary.

In addition, it should be noted that contemplated formulations mayinclude one or more additional pharmaceutically active agents, which maybe present in the same formulation, or be separately made available (ina different type of formulation or the same), or be sold as a kit. Forexample, suitable additional pharmaceutically active agents includevarious steroids (e.g., corticosteroids), agents that stimulate plateletproduction in bone marrow (e.g., Li₂CO₃, folic acid, etc.), antibodies,analgesics, and anti-inflammatory agents.

Contemplated Uses

Contemplated compounds may be particularly useful as therapeutic agentsfor single or combination use in the treatment of (a) nuclearaccident/radiation induced bone and gastrointestinal (GI) damage; (b)chemotherapy induced bone and GI damage; (c) burn inducedthrombocytopenia and GI damage; (d) other causes of thrombocytopenia;(e) other causes of GI damage, including inflammatory bowel diseaseslike Crohn's Disease and ulcerative colitis, as well aspseudomembraneous colitis, (f) free radical-induced lung damage, and (g)cardiovascular diseases.

Consequently, the inventors also contemplate use of the compoundspresented herein for the manufacture of a drug for treatment of (a)nuclear accident/radiation induced bone and GI damage; (b) chemotherapyinduced bone and GI damage; (c) burn induced thrombocytopenia and GIdamage; (d) other causes of thrombocytopenia; (e) other causes of GIdamage, including inflammatory bowel diseases like Crohn's Disease andulcerative colitis, as well as pseudomembraneous colitis, (f) freeradical-induced lung damage, and (g) cardiovascular diseases.

Viewed from another perspective, the inventors also contemplate methodsof treatment of (a) nuclear accident/radiation induced bone and GIdamage; (b) chemotherapy induced bone and GI damage; (c) burn inducedthrombocytopenia and GI damage; (d) other causes of thrombocytopenia;(e) other causes of GI damage, including inflammatory bowel diseaseslike Crohn's Disease and ulcerative colitis, as well aspseudomembraneous colitis, (1) free radical-induced lung damage, and (g)cardiovascular diseases in a human in need thereof in which contemplatedcompounds are administered at a therapeutically effective dosage.

EXPERIMENTS AND EXPERIMENTAL DATA

Materials: Purified bulk of recombinant human IL-11, derived from yeastwas provided by Hangzhou Jiuyuan Gene Engineering Company (Lot#20121005/1006/1007/1008). 7TD1 murine hybridoma cell line, was acquiredfrom DSMZ (No. ACC 23). Paraplatin® injection (generic name:carboplatin) 10 mg/mL (Lot: 5A03935) was manufactured by Bristol-MyersSquibb Company. Trypsin of sequencing grade, modified from bovinepancreas (Cat. No. 11418025001) was purchased from Roche diagnostics.Mouse IL-11 receptor alpha was acquired from MyBioSource, Inc. (Cat. No.MBS553276). CellTiter 96® Aqueous Non-Radioactive Cell ProliferationAssay (MTS) (Cat. No. G5430) was purchased from Promega for 7TD1 cellassay. The DuoSet ELISA development kit for Human IL-11 was purchasedfrom R&D Systems Inc. (Cat. No. DY218). The Purification resin-MacroCapSP (Product code 17-5440-01) was acquired from GE Healthcare LifeSciences. The Precise Tris-Glycine 8-16% polyacrylamide gels werepurchased from Thermo Scientific. Trifluoroacetic acid (Cat. No. 302031)and acetonitril (Cat. No. 34967) for HPLC use were purchased fromSigma-Aldrich.

Monofunctional PEG of various forms with Cat. No. SUNBRIGHT® ME-120TS,ME-200AL, GL2-400TS, ME-050TS, GL4-400AL3, GL2-200AL3, were purchasedfrom NOF Corporation, and Y-PALD-40K was purchased from JenkemTechnology USA. Molecular structures of the PEG reagents were shownabove in Table 1.

Preparation of I12KL/I40KY/I05KL4: 5 mg/mL protein was introduced withmixture of 1 to 2-fold molar ratio of respective PEG reagent(NOF/SUNBRIGHT ME-120TS for I12KL; SUNBRIGHT GL2-400TS for I40KY) and 50mM NaHCO₃ at pH about 8. I05KL4 (PEG reagent: NOF/SUNBRIGHT ME-120TS)was prepared with the same manner except the molar ratio of PEG toprotein was added at 12-fold. The reaction mixture was incubated at roomtemperature for 2 hours, followed by quenching with 2 mM glycine.PEGylated product was isolated using chromatographic purificationprocedures as followed. The PEG molecule was linked to protein by amidebonding.

Preparation of I20NL/I40NY/I20NY/I20NL2/I20NY2/I40NX: 5 mg/mL proteinwas introduced with mixture of 1 to 2-fold molar ratio of respective PEGreagent (NOF/SUNBRIGHT ME-200AL for I20NL and I20NL2; Jenkem/Y-PLAD-40for I40NY; NOF/SUNBRIGHT GL2-200AL3 for I20NY and I20NY2; NOF/SUNBRIGHTGL4-400AL3 for I40NX), 10 mM sodium cyanoborohydride and 50 mM NaH₂PO₄.For conjugating onto two sites, the molar ratio of PEG was added at 3.5to 5.5-fold. The pH was adjusted to about 4.5-5.0. The reaction mixturewas incubated at room temperature for 24 hours, followed by quenchingwith 2 mM glycine. The PEG molecule was linked to protein by a morestable amine bonding. PEGylated product was isolated usingchromatographic purification as followed.

Chromatographic Purification: The pH of protein solution was adjusted to4-5 with 1M acetic acid, followed by centrifugation or filtration toremove particulates. Four volumes of water was introduced. For conjugatecontaining PEG over 20 KDa: The protein solution was loaded onto aMacroCap SP column (1×6 cm) that was equilibrated with buffer Acontaining 20 mM sodium acetate pH 5. The protein was eluted withgradient- or step-elution of buffer B, containing 20 mM sodium acetatepH 5 and 1M NaCl. For conjugate containing PEG below 20 KDa: The proteinsolution was loaded onto a MacroCap SP column (1×6 cm) that wasequilibrated with buffer A containing 20 mM sodium phosphate pH 7. Theprotein was eluted with gradient- or step-elution of buffer B,containing 20 mM sodium phosphate pH 7 and 1M NaCl. A typical finalproduct as analyzed in a SDS PAGE gel can be seen in FIG. 2. Here, theleft lane was loaded with molecular weight markers, and variousPEGylated forms of IL-11 were loaded into the remaining lanes. Note thatI40NY ran at an apparent molecular weight of over 100 Kd, bigger thanthe estimated one of 60 Kd, which is likely due to the Y-shaped of itsPEG moiety. In further particularly preferred aspects, purification ofcontemplated compounds is performed as a one-step purification process,which provides added advantages in a downstream scale-ups.

Purity Check by RP-HPLC: The content of each PEGamer was analyzed byreverse-phase (RP) chromatography employing the UPLC coupled withdiode-array detector-UltiMate 3000 Rapid Separation LC Systems fromThermo Scientific. The chromatographic procedure was carried out using:Column: Acquity C18, 1.7 μm, 2.1×150 mm, 300 Å pore size, equipped witha guard cartridge; Mobile phase A: 0.1% (v/v) TFA in 50% (v/v)acetonitrile; Mobile phase B: 0.1% (v/v) TFA in 95% (v/v) acetonitrile;Flow rate: 0.4 ml/min; Column temperature: 65° C.; Detection: 214 nm;Inject 20 μg and run gradient as in Table 3 below

TABLE 3 Time (min) A % B % 0 100 0 2 100 0 9.9 80 20 9.95 0 100 11.3 0100 11.31 100 0 17.5 100 0

Determination of Protein Content: The protein content was determined bythe UV/Vis microplate and cuvette spectrophotometer—Multiskan GO fromThermo Scientific. Extinction coefficient in units of M⁻¹ cm⁻¹, at 280nm measured in water is 17,990. Alternatively protein concentration isdirectly determined by ultraviolet spectroscopy at wavelength 280 nm,using the absorbency value of 0.944 for a 0.1% (1 mg/ml) solution.Protein quantitation using absorbance at 280 nm measures the absorbanceof aromatic amino acids such as tryptophan and tyrosine, leaving PEGmoiety undetected. As a result, protein concentration by weight statedherein does not contain PEG molecule.

Pharmacokinetics (PK) study in healthy rats: The in vivo manipulationwas carried out in 3 male Sprague-Dawley rats following single doseadministration of contemplated compounds by intravenous or subcutaneousroute at a dosing level of 100-150 μg/kg. Blood samples were collectedin numerous time points in heparin tubes, followed by plasma separationand storage at −20° C. The concentration of immunoreactive IL-11 inplasma samples were determined by the DuoSet ELISA kit for Human IL-11(R&D Systems Inc. Cat. No. DY218). The parameters of pharmacokineticswere yielded by the WinNonlin 5.3 software using non-compartment model.

Pharmacodynamics (PD) study in healthy rats: The pharmacodynamicsassessment was carried out in 4 male Sprague-Dawley rats usingintravenous or subcutaneous administration of respective contemplatedcompounds at dosing strength of 100-150 μg/kg. Blood samples werecollected in numerous time points in heparin tubes, followed by plasmaseparation and storage at −20° C. The blood cell count was carried outon a Cell-DYN 3500 hematology analyzer.

Pharmacodynamics (PD) study in myelosuppressive rats: Thepharmacodynamics assessment was carried out in 4 male Sprague-Dawleyrats, using intravenous administration of carboplatin at 40 mg/kg on Day0 to induce myelosuppression. Contemplated compounds were subcutaneouslyinjected on Day 1 at 150 μg/kg. Blood samples were collected in numeroustime points in heparin tubes, followed by plasma separation and storageat −20° C. The blood cell count was carried out on a Cell-DYN 3500hematology analyzer.

Tryptic mapping: The reaction solution was prepared in 50 mM Tris pH 8.3buffer containing 2 mg/mL protein and 1/50 (W/W) trypsin. Incubated atroom temperature for 6 hours, followed by adding equal volume of 0.2%TFA (trifluoroacetic acid) solution. Any particulate matters wereremoved by centrifuge prior injection onto HPLC. The chromatographicprocedure was carried out using: Column: Zorbax 300 SB-C8, 2.1×150 mm, 5μm, 300 Å pore size; Mobile phase A: 0.1% (v/v) TFA; Mobile phase B:0.1% (v/v) TFA in 95% (v/v) acetonitrile; Flow rate: 0.2 ml/min;Detection: 214 nm; Inject 10 μg and run gradient as in Table 4 below.

TABLE 4 Time (min) A % B % 0 100 0 3 100 0 8 95 5 45 55 45 45.1 0 100 520 100 52.1 100 0 65 100 0The identification of proteolytic peptide was carried out with HPLCcoupled with MS spectrometry (Thermo LCQ Advantage).

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

While it is generally known that PEG can impart the plasma stability ofits conjugates, it is not predictable what attachment type, chainlength, and molecular structure will produce a specific result withrespect to therapeutic effect and/or pharmacologic parameter. In a firstexample, the truncated IL-11 conjugates of various PEG size wereinvestigated with regard to plasma stability in normal rats. Afterintravenous administration, the observed plasma half-life ofun-PEGylated IL-11 was found to be very short, less than 10 minutes ascompared to those (3.5-13.7 hr) of its PEGylated counterparts. Among thelatter, higher molecular-weight PEG imparted greater plasma stability inthe following order: I40KY (13.7 hr) ˜I40NY (8.5 hr) >I20NL (3.8 hr)˜I12KL (3.5 hr). FIG. 3 illustrates the plasma concentration of thevarious forms of PEGylated IL-11 after single intravenousadministration. Each sample was dosed at 100 μg/kg in rats. It wasconcluded that in this example larger or longer PEG chain resulted inlonger serum half-life.

In a second example, pharmacodynamics assessment was carried out withhealthy Sprague-Dawley rats following intravenous route measuringplatelet increment after single intravenous administration at 100 μg/kg.As can be seen, from FIG. 4, 40-KD conjugates (I40NY and I40KY) inducedmore platelet increment (60-75%) than I20NL (50%), where Y representedthe Y-shape PEG and L the linear shape PEG. The results also suggestedmultiple conjugation (I05KL4, 5-KD PEG conjugated on four sites) to beless effective than a single long PEG chain on the N-terminus, asmultiples with shorter PEG conjugates had only limited efficacy at about25% of platelet increment. It was concluded in this example that longerPEG chain on single site resulted in a higher efficacy in the respect ofplatelet induction. This appears to be opposite to the effect ofPEGylation on recombinant human growth hormone.

In a third example, conjugation sites were investigated by trypticmapping, coupled with LC/MS identification of proteolytic peptides. FIG.5 depicts tryptic maps of IL-11 (unconjugated), I40NY, and I40KY, andTable 5 below provides the tryptic peptides.

TABLE 5 Mass Position Peptide # Peptide Sequence 773.9 1-8 T1 GPPPGPPR669.7  9-14 T2 VSPDPR 1217.4 15-25 T3 AELDSTVLLTR 774.9 26-32 T4 SLLADTR798.9 33-39 T5 QLAAQLR 261.3 40-41 T6 DK 3319.8 42-74 T7FPADGDHNLDSLPTLAMSAG ALGALQLPGVLTR 287.4 75-76 T8 LR 950.1 77-84 T9ADLLSYLR 839.0 85-90 T10 HVQWLR 174.2 91-91 T11 R 618. 7 92-98 T12AGGSSLK 1327.5  99-110 T13 TLEPELGTLQAR 402.5 111-113 T14 LDR 400.5114-116 T15 LLR 174.2 117-117 T16 R 860.1 118-124 T17 LQLLMSR 2600.0125-150 T18 LALPQPPPDPPAPPLAPPSS AWGGIR 1914.2 151-168 T19AAHAILGGLHLTLDWAVR 655.9 169-174 T20 GLLLLK 275.3 175-176 T21 TR 131.2177-177 T22 L

Here it can be seen that the peak corresponding to the T1 peptide wasnoticeably reduced in the tryptic maps of I40NY and I40KY. Thisindicates that both conjugates with PEG were linked onto T1 peptides,where the N-terminal amine was the only site for the chemicalconjugation. As a result, I40NY and I40KY were both N-terminally linked,however only differed in chemical bonding with amine bond for I40NY andamide bond for I40KY. Notably, both I40NY and I40KY exhibited similareffects in serum half-life and platelet induction via intravenousadministration.

Since an amine bond is more stable than an amide bond, and yield ofmono-PEGylated product was more homogeneous for selective PEGylationusing reductive amination, in the next study, various N-terminalconjugates were investigated in terms of efficacy in plateletproduction, and were evaluated by their respective associatedside-effect via subcutaneous administration in healthy rats. FIG. 6depicts results of a pharmacodynamics study of six PEGylated IL-11conjugates in rats with subcutaneous administration at 150 μg/kg. IL-11was administered daily for continuous 14 days while PEGylated IL-11 wereinjected once weekly. PEG shape can influence the function of theconjugate. Particularly, the non-linear shape PEG molecule impartsbetter plasma stability and more potency than its linear counterpart. Asdisplayed in FIG. 6, I20NY induced more platelet increment (58-70%) thanI20NL (46-55%), where Y represented the Y-shape PEG and L the linearshape PEG. These results suggested that a Y-shaped PEG had a greatereffectiveness than the linear form of the same molecular weight.However, I40NY (Y-shape) and I40NX (4-arm comb-shape) were comparable inplatelet production as both increased up to about 65-70%, suggesting theinfluence of PEG shape became saturated when the PEG size was about orover 40 KD. Notably, double PEGylation of the same PEG length reducedthe in vivo efficacy, as I20NL2 (linear PEG onto two sites) and I20NY2(Y-shaped PEG onto two sites) had lower platelet production than theirsingle PEGylated counterparts. It was therefore concluded that I40NY,I40NX and I20NY exerted higher efficacy among various N-terminalconjugated IL-11s. Moreover, it was noted that the effect of secondadministration was somehow down-regulated for smaller PEG conjugatessuch as 20-KD PEG regardless of number of conjugation site. As a result,I40NY and I40NX were unexpectedly effective compounds with desirablebiological properties and relatively moderate adverse effects(especially plasma expansion). Moreover, the biological data furthersuggest that the so modified IL-11 compounds can be administered lessfrequently, and most preferably twice weekly, once weekly, or even less.Such schedule is particularly relevant where contemplated compounds areemployed in the treatment of thrombocytopenia in a large population(e.g., exposed to radiologic exposure).

The inventors also studied the side-effect associated with IL-11conjugates in healthy rats. Hematocrit status is usually used as amarker for assessment of side-effect in clinical use of IL-11, becausepatients might experience dilutional anemia due to plasma expansion. Inthe animal study, IL-11 was administered subcutaneously for continuous14 days at 150 μg/kg while PEGylated IL-11 were injected once weekly atthe same dose. As shown in FIG. 7, all drugs resulted in decreasedhematocrit but I40NY was found to have less reduction while maintainedhigher activity than the rest of PEGylated conjugates. The mitigateddilutional anemia of dosing with I40NY is more prominent when comparedin the same chart with other individual animal experiments usingconjugated and unconjugated IL-11s. A correlation between plateletproduction and side-effect as manifested by reduction of hematocrit wasestablished in FIG. 8, suggesting a trend of intensifying side-effectalong with the increasing and dose-dependent efficacy when variousmodified and unmodified IL-11s were plotted in the chart. I40NYs, atdifferent doses, were distinctly located on the right upper side of thetrend, indicating less plasma expansion than some other compounds andunmodified IL-11 on the basis of comparable efficacy. In terms ofproduct characterization, the inventors characterized physicochemicaland pharmacological properties of a preferred compound, I40NY.

Cell-based Assay for IL-11 Conjugates: The biological activities ofconjugated IL-11 were tested in a cell proliferation assay, using 7TD1cell-line (DSMZ, Germany) In brief, 7TD1 cells at 4,000 cells per wellwere grown at 37° C. in a humidified atmosphere of 5% CO₂ in response todifferent IL-11 concentrations for two days in the presence of 2 μg/mLmurine IL-11 receptor (MyBioSource, USA, MBS553276)(Biochem. J.,318:489-495). After addition of MTS, the EC50 of dose response curve wasdetermined by plotting the absorbance of 490 nm on the y-axis againstIL-11 concentrations on the y-axis, by fitting sigmoid dose-responsecurves with GraphPad software Prism 6. Before animal studies, thebiological activities of newly synthesized conjugates were tested in acell proliferation assay, using 7TD1 cell line. Not all PEGylationpreparations gave similar products, and the actual product formationdepends on the amino acid residues of the IL-11 being conjugated, and onthe size and the shape of the PEG molecule used. 7TD1 cells grew inresponse to different conjugate concentrations. After addition ofdevelopers, whose chemical signal has a linear relationship with thecell number, absorbance at 490 nm was read in an ELISA plate-reader. Theresults were shown in FIG. 9.

Due to the steric hindrance of the PEG moiety, all the conjugatesexpectedly showed a reduction in bioactivity in the cell-based assay ascompared to that of the un-PEGylated IL-11 with the following order ofpotency: IL-11 (100%) >I20NL, I20NY (both about 16%) >I40NY, I40NX (both11%) >I40KY (6%) >I20NY2 (3%). Notably, the steric interference is adominant factor in determining biological activity of PEGylatedconjugates and can be seen as a drastic decrease in bioactivity of theconjugate whose overall PEG moiety is bigger than 20 KDa. From somecell-based studies, there was a report showing that a small carbohydrateattachment at the non-core region of the IL-11 molecule, such asN-terminal sequence, enhanced the biological activity when compared withthose conjugates at other attachment sites (J. Biol. Chem. Vol 286, No.10, pp 8085-810

093), which was consistent with less reduction of bioactivity by PEGmolecule at the N-terminal sequence of IL-11. Although the in vitrobioactivity of I40NY retained only about 11% of native IL-11, in vivoefficacy was beneficially affected and could not be predicted from thein vitro bioactivity data. Table 6 below schematically illustratesbioactivity ratios of various compounds relative to unmodified IL-11

TABLE 6 Bioactivity ratio to Identification unmodified IL-11 IL-11 1I20NL 0.13 I20NY 0.16 I20NY2 0.03 I40NY 0.11 I40NX 0.11 I40KY 0.06

Chemical modification of proteins with PEG is an established technologyand has been applied to biopharmaceutical industry to enhance thesolubility and physical-chemical stability of proteins. While thischemical reaction is easy to carry out, it often results in a complexmixtures of different PEGylated forms, containing PEGamer and positionalisomers. Multiple chromatographic purifications steps are employed toisolate product with high recovery. To develop a commercially viableprocess in terms of cost and yield, many factors such as proteinconcentration, quality of PEG, protein/PEG ratio, reaction temperature,and buffer pH, as well as purification process, are required to beoptimized.

I40NY was constructed by conjugation with a Y-shaped polyethylene glycolchain on amines forming stable amine bond, with relatively highselectivity to the N-terminal amine driven by conjugation chemistry(reductive amination of aldehyde coupling group in PEG moiety). I40KY onthe other hand was conjugated using a functionalized NHS reagent at pH 8on accessible amines forming the corresponding amide bonds. Morespecifically, I40NY is the mono-PEGylated IL-11 produced withsite-specific reaction under acidic conditions because functionalizedaldehyde is largely selective for the N-terminal α-amine, whose pKa islower compared to other nucleophiles. PEG to protein ratio, reactionconcentration, pH and kinetics were investigated in the conjugationreaction. Reactions were taken place for 24 hours at room temperature(22-27° C.) in small scale at about 0.05-0.5 mL in volume in thepresence of 10 mM sodium cyanoborohydride. Yield of each reaction underinvestigation was determined by RP-UPLC. Using different pH for selectedreactions, optimum conjugation yield was at pH 4.5-5.5. In addition, theinventors noted that the concentration of reactants played an importantfactor in product yield and discovered that conjugation with IL-11 atconcentrations larger than 5 mg/mL were optimal. Likewise, the PEG toprotein ratio and conjugation kinetics of the reaction with 5 mg/mLprotein at room temperature in the presence of reducing agent wereinvestigated and suggested an optimum molar ratio of 2 for PEG toprotein, and reaction extension to 16 hours sufficient formono-PEGylation.

Purification of PEGylated protein usually employs ion-exchangechromatography in large-scale preparation. However a satisfactoryresolution to separate the mono-PEGylated from the oligo-PEGylated isnot achievable when conventional ion-exchanger is loaded with reactionproduct at a loading capacity as little as 1 mg/mL resin. The lowcapacity of this resin often limits its application for larger scaleproduction. To isolate N-terminally mono-PEGylated IL-11 at high purity,various cation-exchange resins were tested. Notably, high porosityresins (e.g., MacroCap SP from GE Healthcare Life Sciences) providedhigh capacity with retained resolution, and offered high purity andyield of mono-PEGylated targets at high load conditions. Thepurification process was demonstrated with a batch size of 400 mg IL-11,prepared in 5 mg/mL in sodium phosphate pH 4.5-5 buffer containing 2molar ratio of aldehyde-activated 40-KD Y-shaped PEG reagent in thepresence of 10 mM sodium cyanoborohydride. The reaction solution wasquenched by adding 2 M glycine followed by dilution with 4× volume ofdeionized water. After filtration through a 0.2 μm membrane, theresulting crude was loaded onto a MacroCap SP column (2.6 (diameter)×10(height) cm at a loading capacity of about 7.5 mg/mL resin). Aftercharging, the column was washed with 20 mM sodium acetate pH 5 bufferover 10 column volume, followed by additional wash with 20 mM sodiumacetate pH 5 buffer containing 0.1 M NaCl over 20 column volume. Theproduct was then eluted with 20 mM sodium acetate pH 5 buffer containing0.3 M NaCl. The overall yield of isolating I40NY was 26.6%. The productpurity of I40NY was examined by SDS-PAGE and reverse-phase HPLC, andFIG. 10 shows purity of I40NY on a silver-stained SDS-PAGE gel withquantities of I40NY as indicated above the lanes. The purity ofmono-PEGylated IL-11 was larger than 93% as determined by C18-HPLC asthe chromatogram displayed in FIG. 11.

To determine the pharmacokinetic parameters of I40NY via subcutaneousroute, 3 male Sprague-Dawley rats were injected with 0.15 mg/kg ofPEGylated IL-11 via single subcutaneous administration. FIG. 12displayed the plasma concentration of immunoreactive IL-11 in rats aftersingle subcutaneous administration. Plasma concentration of conjugatedIL-11 achieved a maximal level at about 12 hr and remained effectiveover 50 hr after administration. On the contrary, the recombinant humanIL-11 reached a maximal concentration at about 2 hr, and was clearedfrom the circulation blood stream rapidly as the elimination half-lifein plasma was about 1.3 hr. The pharmacokinetic parameters of I40NY viasubcutaneous route were summarized in Table 7 below.

TABLE 7 unit I40NY IL-11 T_(1/2), terminal half-life hr 18.6 1.1T_(max), time to maximal concentration hr 12 2 C_(max), maximal plasmaconcentration ng/mL 142 147 AUC_(all), area under curve Hr*ng/mL 3947700 AUC_(inf), area under curve to infinity Hr*ng/mL 4421 701 Vz,relative volume of distribution mL/kg 909 347 Cl, relative clearancemL/hr/Kg 33.9 214 MRT, mean residence time hr 27.2 3.7

The secondary structure of I40NY was investigated using circulardichroism. In the chromatogram of a circular dichroism analyzed in thefar-UV region, the inventors demonstrated that I40NY maintained the samesecondary structure as its unconjugated counterpart, as can be seen fromboth spectra superimposed in FIG. 13. Moreover the thermal stability ofI40NY was demonstrated by circular dichroism by measuring the change oftheir secondary structures (mean residue ellipticity) in response tothermal stress. FIG. 14 indicated the structural change being less forI40NY in response to temperature increment.

The effectiveness of I40NY in myelosuppressive rats was alsodemonstrated in carboplatin-treated rats. Male Sprague-Dawley rats wereinjected with 40 mg/kg of carboplatin via intravenous administration toinduce damaged function of bone marrow leading to thrombocytopenia.Medical intervention using daily injection (consecutive 7 days) of IL-11or single dose of I40NY at the same 0.15 mg/kg dosage was subcutaneouslyadministered immediately after 24 hours of carboplatin treatment. Theplatelet level was displayed in FIG. 15. Without treatment, subjectsexperienced about two days of severe thrombocytopenia (less than ⅓ ofnormal platelet count), suggesting the untreated having a high risk oflife threatening internal bleeding. The efficacy of IL-11 treatment wasmarginal as the nadir of daily dosing was very close to the threshold ofsevere thrombocytopenia. Single dose of I40NY, on the contrary, not onlyprevented the occurrence of severe thrombocytopenia but also acceleratedthe recovery of platelet levels, as the platelet count returning to theinitial number was 1.3 days earlier than the other two groups.

Meanwhile, the side effects as manifested on the reduction of hematocritwas also investigated in the myelosuppressive model. In FIG. 16,treating with IL-11s caused hematocrit reduction in a rapid mannercompared to the untreated group. However single dose of I40NY alleviatedthe nadir, suggesting less intensified side-effect than daily dosingwith IL-11. Thus, it should be appreciated that I40NY has proveneffective in preventing severe thrombocytopenia induced by chemotherapy,while ameliorating the syndrome of plasma expansion.

Further comparative data between I40NY and another form of a PEGylatedIL-11 (as described in U.S. Pat. No. 8,133,480, data not shown) revealthat contemplated compounds, and especially I20NY and I40NY hadsignificantly enhanced in vivo effectiveness and reduced syndrome ofside-effect as compared to the other form of PEGylated IL-11 asdescribed in the '480 patent.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the scope of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of preparing an interleukin 11 (IL-11)conjugate, comprising: contacting an IL-11 polypeptide chain with a PEGmoiety comprising an amine-selective reactive group at an equimolar to2-fold molar excess of the PEG moiety relative to the IL-11 peptide a pHof from 4.5 to 5.0 and ambient temperature for at least 24 hours,wherein the PEG moiety has an average molecular weight of 10 kD to 50 kDand has at least two branches; and collecting a resulting IL-11conjugate preparation, wherein the resulting IL-11 conjugate preparationcomprises a monosubstituted IL-11 conjugate at a purity of greater than93%, wherein the IL-11 polypeptide chain is a human IL-11 or truncatedhuman IL-11 polypeptide chain.
 2. The method of claim 1 wherein theIL-11 polypeptide chain is a human IL-11 polypeptide chain.
 3. Themethod of claim 1 wherein the IL-11 polypeptide chain is truncated bydeletion of an N-terminal proline.
 4. The method of claim 1 wherein theIL-11 polypeptide chain has a sequence according to SEQ ID NO:1.
 5. Themethod of claim 1 wherein the PEG moiety has an average molecular weightof 20 kD or 40 kD.
 6. The method of claim 1 wherein the PEG moiety has aY shape.
 7. The method of claim 1 wherein the PEG moiety is covalentlybound to the Il-11 peptide's N-terminus via an amine bond.
 8. The methodof claim 1, wherein the amine-selective reactive group is an aldehydegroup.